GITR (also known as TNFRSF18) is a co-stimulatory member of the TNF receptor superfamily. Expression of GITR has been observed predominantly on T cells, NK cells, B cells, and to a lesser extent on some other hematopoietic cell types. GITR exhibits low expression in resting T and NK cells, but constitutively high level expression in CD4+Foxp3+ regulatory T cells (Tregs). In vitro or in vivo engagement of GITR by GITR ligand or agonist anti-GITR antibodies causes the expansion of CD4+ and CD8+ T cells and improves the resistance of T cells to suppression by Tregs.
Preclinical evidence suggests that inducing GITR signaling can enhance the activation of effector T cells (Teffs) and reduce the activity of Tregs in experimental tumours. Dosing with agonist anti-GITR mAbs leads to enhanced endogenous Teff responses, reduction in the frequency of Tregs in the tumour microenvironment, and subsequent tumor rejection in multiple murine tumor models. Hence, anti-GITR mAbs have the potential to act as immunotherapeutic agents in cancer and other settings, and also to amplify the effectiveness of currently established cancer immunotherapies.
The majority of currently approved antibody therapeutics are derived from immunized rodents. Most of those antibodies have undergone a process known as “humanization”, via the “grafting” of murine CDRs into human v-gene framework sequences (see Nelson et al., 2010, Nat Rev Drug Discov 9: 767-774). This process is often inaccurate and leads to a reduction in target binding affinity of the resulting antibody. To return the binding affinity of the original antibody, murine residues are usually introduced at key positions in the variable domain frameworks of the grafted v-domains (also known as “back-mutations”).
While antibodies humanized via CDR grafting and back mutations have been shown to induce lower immune response rates in the clinic in comparison to those with fully murine v-domains, antibodies humanized using this basic grafting method still carry significant clinical development risks due to the potential physical instability and immunogenicity motifs still housed in the grafted CDR loops. As animal testing of protein immunogenicity is often non-predictive of immune responses in man, antibody engineering for therapeutic use focuses on minimizing predicted human T-cell epitope content, non-human germline amino acid content and aggregation potential in the purified protein.
The ideal humanized agonistic anti-GITR antibody would therefore have as many identical residues as possible in the v-domains to those found in both the frameworks and CDRs of well-characterized human germline sequences. Townsend et al. (2015; PNAS 112: 15354-15359) describe a method for generating antibodies in which CDRs derived from rat, rabbit and mouse antibodies were grafted into preferred human frameworks and then subject to a human germ-lining approach termed “Augmented Binary Substitution”. Although the approach demonstrated a fundamental plasticity in the original antibody paratopes, in the absence of highly accurate antibody-antigen co-crystal structural data, it is still not possible to reliably predict which individual residues in the CDR loops of any given antibody can be converted to human germline, and in what combination.
CDR germ-lining is thus a complex, multifactorial problem, as multiple functional properties of the molecule should preferably be maintained, including in this instance: target binding specificity, affinity to GITR from both human and animal test species (e.g. cynomolgus monkey, also known as the crab-eating macaque, i.e. Macaca fascicularis), v-domain biophysical stability and/or IgG expression yield. Antibody engineering studies have shown that the even single residue positions in key CDRs can have dramatic effects on all of these desired molecular properties.
WO2006/105021 describes an agonistic murine anti-GITR IgG molecule termed “6C8”, and also the preparation of humanized forms of 6C8. Those humanized forms of 6C8 were produced using classical humanization techniques, i.e. by grafting of Kabat-defined murine CDRs into human heavy and light chain framework sequences and some of the human framework residues back-mutated to the correspondingly positioned 6C8 murine residues. Only one amino acid modification to one of the Kabat-defined murine CDRs of 6C8 is described in WO2006/105021, to modify a potential glycosylation site. For reasons noted above, such humanized forms of 6C8 described in WO2006/105021 are not ideal.
The present invention provides a number of optimized anti-GITR antibodies and medical uses thereof.